ونحن نشكر أعضاء فريق العمل لدينا، Blache MC خاصة لتقديم المساعدة التقنية، وكذلك M Irla Imhof وباء، ولما قدموه من دعم ومناقشات مثمرة. استنساخ الأرقام لالانكماش والحبس مجاملة من طرق الطبيعة. هذا المشروع يتم بدعم من المنح المقدمة من المؤسسات CNRS، INSERM وجامعة مرسيليا، ومنح محددة من منطقة بروفانس ألب كوت--d&#39;Azur، المعهد الوطني للسرطان دو، الوكالة الوطنية دي لا بحوث (ANR-PCVI-08- 0034-02، ANR بلان 2010 1214 01) ومؤسسة بور لا بحوث ميديكال (إيكيب labélisée FRM-2009). ويدعم VR من قبل زمالة من السرطان الوطني الفرنسي جنيه كونتر.

<ol>
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الإعلان عن أي تضارب في المصالح.

في واحدة تتبع الجسيمات، بجانب الجوانب الخلية والمجهري، وتحليل يمثل جزءا كبيرا من العمل. هذا ويتناول الخوارزمية المستخدمة لتنفيذ المهام الرئيسية الثلاث: كشف وتقدير وإعادة قمم فوق كل إطار. لكن الجانب يترتب على ذلك من هذا العمل تكمن في وضع الخوارزمية نفسها، والتي قد تح...

<table border="1" style=";text-align:right;direction:rtl"><tbody><tr><td> الكاشف </td><td> شركة </td><td> فهرس العدد </td><td> كمية </td></tr><tr><td> كوس-7 خط الخلية </td><td> ATCC </td><td> CRL-1651 </td><td> 5000 خلايا / جيد </td></tr><tr><td> HBSS دون كا 2 + </td><td> GIBCO </td><td> 14175 </td><td> 1 مل </td></tr><tr><td> 0.05٪ التربسين EDTA </td><td> GIBCO </td><td> 25300 </td><td> 1 مل </td></tr><tr><td> 8-جيدا مختبر تيك </td><td> نونك </td><td> 155441 </td><td> 1 </td></tr><tr><td> QDot-605 streptavidin </td><td> إينفيتروجن </td><td> Q10101MP </td><td> 20 ملي مول </td></tr><tr><td colspan="4"> المعقدة البيروكسيديز فاب (لتخليق فاب، أنظر المرجع 21) </td></tr><tr><td> القوات المسلحة البوروندية من 108 خريطة موقع </td><td> ATCC </td><td> HB-9764 </td><td> 200 ميكروغرام </td></tr><tr><td> NHS-بيوتين<html></td><td> الحرارية العلمية </td><td> 21435 </td><td> 18.5 ميكروغرام </td></tr><tr><td colspan="4"> إكمال المتوسطة </td></tr><tr><td> DMEM </td><td> GIBCO </td><td> 41965 </td><td> 500 مل </td></tr><tr><td> الأبقار مصل الجنين </td><td> SIGMA </td><td> F7524 </td><td> 50 مل </td></tr><tr><td> L-الجلوتامين </td><td> GIBCO </td><td> 25030 </td><td> 5 مل </td></tr><tr><td> HEPES </td><td> GIBCO </td><td> 15630 </td><td> 5 مل </td></tr><tr><td> الصوديوم البيروفات </td><td> GIBCO </td><td> 11360 </td><td> 5 مل </td></tr><tr><td colspan="4"> التصوير المتوسطة </td></tr><tr><td> HBSS مع كا 2 + </td><td> GIBCO </td><td> 14025 </td><td> 25 مل </td></tr><tr><td> HEPES </td><td> GIBCO </td><td> 15630 </td><td> 250 ميكرولتر </td></tr></tbody></table>1 &quot;&gt; <tbody><tr><td> معدات </td><td> شركة </td><td> مرجع </td></tr><tr><td> المقلوب المجهر </td><td> نيكون </td><td> كسوف TE2000U </td></tr><tr><td> مصباح فلوري </td><td> نيكون </td><td> Intensilight C-HGFIE </td></tr><tr><td> 1.3 NA 100X هدف </td><td> نيكون </td><td> تخطط شركة فلور 1.30 </td></tr><tr><td> 1.49 هدف NA 100X </td><td> نيكون </td><td> APO TIRF 1.49 </td></tr><tr><td> كاميرا </td><td> روبر العلمية </td><td> تتالي 512 B </td></tr><tr><td> Thermostated مربع </td><td> حياة التصوير خدمات </td><td> مربع </td></tr></tbody></table> ملحق: سيناريو سبيل المثال من تحليل تكميلي MTT وظيفة MTT_example (FILE_NAME) أمثلة٪٪٪ الأساسية التي تبين كيفية استرداد نتائج الإخراج MTT ٪٪٪ لرسم كل تراكه وبناء الرسم البياني ٪٪٪ من كثافة مضان إذا nargin &lt;1٪ لا FILE_NAME المقدمة؟ الملفات = دير (&#39;* STK.&#39;)؛ إذا isempty (ملفات)، DISP (&#39;لا توجد بيانات في المسار الحالي&#39;)، والعودة، نهاية . FILE_NAME = الملفات (1) الاسم؛ الافتراضية٪: أولا ملف STK DISP ([&#39;باستخدام&#39; FILE_NAME &#39;افتراضيا&#39;]) نهاية file_param = [FILE_NAME &quot;_tab_param.dat &#39;]؛ إخراج الملف٪ بيانات٪ الحمولة استخدام القرص المضغوط (&#39;output23&#39;)٪ أو (&#39;output22&#39;)، وفقا لإصدار تنويه٪: الإصدار 2.2 يولد سوى 7 معلمات، أضيف٪ معلمة إضافية، والضوضاء، في الإصدار 2.3 ٪ لقراءة جميع المعلمات في آن واحد، في جدول واحد tab_param٪ = fread_all_param (file_param)؛ تا٪b_i = tab_param (2:08: نهاية، :)؛ tab_j = ... ٪ لقراءة جميع المعلمات (باستثناء frame_number) في جداول منفصلة ٪ [tab_i، tab_j، tab_alpha، tab_radius، tab_offset، tab_blk، tab_noise] = fread_all_data_spt (file_param)؛ tab_i = fread_data_spt (file_param، 3)؛ مؤشر٪ هي 3 لأن يتم تجاهل رقم التتبع ورقم الإطار، وغير مفيدة،! tab_j = fread_data_spt (file_param، 4)؛ tab_alpha = fread_data_spt (file_param، 5)؛ tab_blk = fread_data_spt (file_param، 8)؛ ٪٪ خلال حلقة آثار N_traces = حجم (tab_i، 1)؛ الجداول٪ من خطوط N_traces بواسطة colums N_frames لitrc = 1: N_traces No_blk_index = tab_blk (itrc، :)&gt; 0؛٪ خطوات امض غير فقط مؤامرة (tab_i (itrc، No_blk_index)، tab_j (itrc، No_blk_index)) &amp; ملاحظة:س؛ xlabel (&#39;ط (بكسل)&#39;)، ylabel (&#39;ي (بكسل)&#39;) لقب ([num2str &#39;تتبع #&#39; (itrc)]) DISP (&#39;الرجاء ضرب أي مفتاح لتتبع المقبل)، وقفة نهاية رسم بياني٪ Fluo N_datapoints = SUM (tab_blk (:)&gt; 0)؛٪ خطوات غير وامض فقط تاريخ (tab_alpha (tab_blk&gt; 0)، 2 * الجذر التربيعي (N_datapoints))٪ باستخدام 2sqrt (N) صناديق xlabel (&#39;كثافة (الاتحاد الافريقي)&#39;)، ylabel (&#39;ظهور&#39;) عنوان (&#39;رسم بياني لكثافة الجسيمات مضان&#39;)

mapping molecular diffusion in the plasma membrane by multiple-target tracing (mtt)

هدفنا هو الحصول على وصف شامل لعمليات الجزيئية التي تحدث في الأغشية الخلوية في الوظائف البيولوجية المختلفة. ونحن نهدف إلى تحديد خصائص منظمة معقدة وديناميكية من غشاء البلازما في مستوى واحد جزيء، من خلال تطوير أدوات تحليلية واحدة مخصصة لتتبع الجسيمات (SPT) في كثافة عالية: الهدف متعددة للبحث عن المفقودين (MTT) 1. واحدة جزيء videomicroscopy، وتقدم ميلي ثانية واحدة وقرار النانومترية 1-11، يسمح لتمثيل مفصل لمنظمة غشاء 12-14 بدقة عن طريق تعيين واصفات مثل مستقبلات الخلايا التوطين، والحبس، والتنقل أو التفاعلات. نحن النظر SPT، على حد سواء بالتجربة وحسابيا. وشملت الجوانب التجريبية تحسين الإعداد ووسم الخلية، مع التركيز بشكل خاص على الوصول إلى كثافة العلامات أعلى مستوى ممكن، من أجل توفير لقطة الحيوية للديناميات الجزيئية 1ق يحدث داخل غشاء. المعنية بقضايا حسابي كل خطوة تستخدم لإعادة بناء مسارات: قمم الكشف، وتقدير وإعادة الاتصال، التي تناولتها وسائل محددة من تحليل الصور 15،16. تنفيذ الانكماش بعد اكتشاف يسمح قمم انقاذ مخبأة في البداية من قبل القمم، أقوى المجاورة. من المذكرة، وتحسين الكشف يؤثر تأثيرا مباشرا لإعادة الاتصال، عن طريق تقليل الفجوات في المسارات. وقد تم تقييم العروض باستخدام مونتي كارلو محاكاة لكثافة العلامات المختلفة والقيم الضجيج، والتي تمثل عادة القيود الرئيسيتين لقياسات موازية في القرار المكانية العالية. يمكن للدقة النانومترية 17 التي تم الحصول عليها عن الجزيئات واحدة، وذلك باستخدام إما المتعاقبة على / قبالة البصريات photoswitching أو غير الخطية، وتقديم الملاحظات شاملة. هذا هو أساس أساليب nanoscopy 17 مثل STORM 18، 19،20 النخيل، RESOLFT 21 أو STED 22،23، مبادرة الخوذ البيضاءقد الفصل غالبا ما تتطلب عينات التصوير الثابت. المهمة المركزية هي الكشف وتقدير من الحيود محدودة القمم الصادرة عن جزيئات واحدة. وبالتالي، توفير افتراضات كافية مثل التعامل مع دقة ثابت الموضعية بدلا من الحركة البراونية، هي مناسبة بشكل مباشر MTT لتحاليل نانوية. وعلاوة على ذلك، يمكن أن تستخدم أساسا MTT بأي مقياس: ليس فقط لجزيئات، ولكن أيضا لخلايا أو الحيوانات، وعلى سبيل المثال. وبالتالي، MTT هو خوارزمية تتبع القوية التي تجد تطبيقات في موازين الجزيئي والخلوي.

Watch this Scientific Journal Video about Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT at JoVE.com

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

متعددة للبحث عن المفقودين الهدف هو خوارزمية محلية الصنع وضعت لتتبع الجزيئات بشكل فردي وصفت داخل الغشاء البلازمي للخلايا الحية. كشف بكفاءة، وتقدير وتعقب جزيئات مع مرور الوقت في كثافة عالية توفر سهل الاستعمال، وأداة شاملة للتحقيق في ديناميات غشاء النانو.

رسم خرائط الانتشار الجزيئي في غشاء البلازما من قبل متعددة الهدف، للبحث عن المفقودين (MTT)

Our goal is to obtain a comprehensive description of molecular processes occurring at cellular membranes in different biological functions. We aim at ...

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Biology

Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing (MTT)

17.1K Views.  Parc scientifique de Luminy. متعددة للبحث عن المفقودين الهدف هو خوارزمية محلية الصنع وضعت لتتبع الجزيئات بشكل فردي وصفت داخل الغشاء البلازمي للخلايا الحية. كشف بكفاءة، وتقدير وتعقب جزيئات مع مرور الوقت في كثافة عالية توفر سهل الاستعمال، وأداة شاملة للتحقيق في ديناميات غشاء النانو.

Video: Mapping Molecular Diffusion in the Plasma Membrane by Multiple-Target Tracing MTT

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

قياس الأدنى غشاء البلازما والعالمية حيوية الكالسيوم بين الخلايا في الخلايا النجمية

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Measuring Plasma Membrane Protein Endocytic Rates by Reversible Biotinylation

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins is responsible for a variety of key cellular events, including nutrient delivery, cellular excitability, and chemical signaling.  Many plasma membrane proteins are dynamically regulated by endocytic trafficking, which modulates protein function by altering protein surface expression.  The mechanisms that facilitate protein endocytosis are complex and are not fully understood for many membrane proteins.  In order to fully understand the mechanisms that control the endocytic trafficking of a given protein, it is critical that the protein s endocytic rate be precisely measured.  For many receptors, direct endocytic rate measurements are frequently achieved utilizing labeled receptor ligands.  However, for many classes of membrane proteins, such as transporters, pumps and ion channels, there is no convenient ligand that can be used to measure the endocytic rate.  In the present report, we describe a reversible biotinylation method that we employ to measure the dopamine transporter (DAT) endocytic rate.  This method provides a straightforward approach to measuring internalization rates, and can be easily employed for trafficking studies of most membrane proteins.

Regulated endocytosis governs the cell surface expression levels of the majority of membrane proteins. Here we utilize reducible, membrane impermeant biotinylation reagents to measure the endocytic rate of the dopamine transporter (DAT), a polytopic membrane protein. The method facilitates a straightforward approach to measuring the endocytic rate of most plasma membrane proteins.

قياس معدلات بلازما بروتين الغشاء التقامي بواسطة Biotinylation عكسية

Plasma membrane proteins are a large, diverse group of proteins comprised of receptors, ion channels, transporters and pumps.  Activity of these proteins ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

قياس الكمي GLUT4 إزفاء إلى غشاء البلازما التي التدفق الخلوي

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

بلورة البروتينات في الغشاء Mesophases Lipidic

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein

Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor's vitamin A uptake mechanism.

Here we describe an optimized technique to produce high-quality vitamin A/RBP complex and two real-time monitoring techniques to study vitamin A transport by STRA6, the RBP receptor.

في الوقت الحقيقي تحليلات النقل الريتينول بواسطة مستقبلات البروتين غشاء البلازما الريتينول ملزم

Vitamin A is essential for vision and the growth/differentiation of almost all human organs.  Plasma retinol binding protein (RBP) is the principle and ...

Easy Measurement of Diffusion Coefficients of EGFP-tagged Plasma Membrane Proteins Using k-Space Image Correlation Spectroscopy

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new insights to plasma membrane protein function and regulation. Recently, k-Space Image Correlation Spectroscopy (kICS)1&#160;was developed to enable routine measurements of diffusion coefficients directly from images of fluorescently tagged plasma membrane proteins, that avoided systematic biases introduced by probe photophysics. Although the theoretical basis for the analysis is complex, the method can be implemented by nonexperts using a freely available code to measure diffusion coefficients of proteins. kICS calculates a time correlation function from a fluorescence microscopy image stack after Fourier transformation of each image to reciprocal (k-) space. Subsequently, circular averaging, natural logarithm transform and linear fits to the correlation function yields the diffusion coefficient. This paper provides a step-by-step guide to the image analysis and measurement of diffusion coefficients via kICS.
First, a high frame rate image sequence of a fluorescently labeled plasma membrane protein is acquired using a fluorescence microscope. Then, a region of interest (ROI) avoiding intracellular organelles, moving vesicles or protruding membrane regions is selected. The ROI stack is imported into a freely available code and several defined parameters (see Method section) are set for kICS analysis. The program then generates a &#34;slope of slopes&#34; plot from the k-space time correlation functions, and the diffusion coefficient is calculated from the slope of the plot. Below is a step-by-step kICS procedure to measure the diffusion coefficient of a membrane protein using the renal water channel aquaporin-3 tagged with EGFP as a canonical example.

This paper provides a step by step guide to the fluctuation analysis technique k-Space Image Correlation Spectroscopy (kICS) for measuring diffusion coefficients of fluorescently labeled plasma membrane proteins in live mammalian cells.

من السهل قياس إنتشار معاملات EGFP الموسومة البروتينات عن طريق غشاء البلازما ك الفضاء صورة الارتباط الطيفي

Lateral diffusion and compartmentalization of plasma membrane proteins are tightly regulated in cells and thus, studying these processes will reveal new ...

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

التصوير البلازما غشاء التشويهات مع pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...

Simultaneous Measurement of Mitochondrial Calcium and Mitochondrial Membrane Potential in Live Cells by Fluorescent Microscopy

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ concentration. To do this, mitochondria utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester Ca2+. The influx of Ca2+ into the mitochondria stimulates three rate-limiting dehydrogenases of the citric acid cycle, increasing electron transfer through the oxidative phosphorylation (OXPHOS) complexes. This stimulation maintains &#916;&#936;m, which is temporarily dissipated as the positive calcium ions cross the mitochondrial inner membrane into the mitochondrial matrix.
We describe here a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using confocal microscopy. By permeabilizing the cells, mitochondrial Ca2+ can be measured using the fluorescent Ca2+ indicator Fluo-4, AM, with measurement of &#916;&#936;m using the fluorescent dye tetramethylrhodamine, methyl ester, perchlorate (TMRM). The benefit of this system is that there is very little spectral overlap between the fluorescent dyes, allowing accurate measurement of mitochondrial Ca2+ and &#916;&#936;m simultaneously. Using the sequential addition of Ca2+ aliquots, mitochondrial Ca2+ uptake can be monitored, and the concentration at which Ca2+ induces mitochondrial membrane permeability transition and the loss of &#916;&#936;m determined.

Mitochondria can utilize the electrochemical potential across their inner membrane (&#916;&#936;m) to sequester calcium (Ca2+), allowing them to shape cytosolic Ca2+ signaling within the cell. We describe a method for simultaneously measuring mitochondria Ca2+ uptake and &#916;&#936;m in live cells using fluorescent dyes and confocal microscopy.

قياس وقت واحد من الميتوكوندريا الكالسيوم والميتوكوندريا غشاء المحتملة في الخلايا الحية التي كتبها نيون الميكروسكوب

Apart from their essential role in generating ATP, mitochondria also act as local calcium (Ca2+) buffers to tightly regulate intracellular Ca2+ ...

Determination of Plasma Membrane Partitioning for Peripherally-associated Proteins

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein using the profiles of fluorescence intensity across the plasma membrane. Measured fluorescence profiles are fitted by a model for membrane and cytoplasm fluorescence distribution along a line applied perpendicularly to the cell periphery. This model is constructed from the fluorescence intensity values in reference cells expressing a fluorescently-tagged marker for cytoplasm and with FM 4-64-labeled plasma membrane. The method can be applied to various cell types and organisms; however, only plasma membranes of non-neighboring cells can be evaluated. This fast microscopy-based method is suitable for experiments, where subtle and dynamic changes of plasma membrane-associated markers are expected and need to be quantified, e.g., in the analysis of mutant versions of proteins, inhibitor treatments, and signal transduction observations. The method is implemented in a multi-platform R package that is coupled with an ImageJ macro that serves as a user-friendly interface.

Here, we present a protocol to perform a quantitative analysis of the level of plasma-membrane association for fluorescently-tagged peripherally-associated protein. The method is based on the computational decomposition of membrane and cytoplasmic component of signal observed in cells labeled with plasma membrane fluorescent marker.

تحديد التقسيم غشاء البلازما للبروتينات المرتبطة محيطيا

This method provides a fast approach for the determination of plasma membrane partitioning of any fluorescently-tagged peripherally-associated protein ...

Characterization of MLKL-mediated Plasma Membrane Rupture in Necroptosis

Necroptosis is a programmed cell death pathway triggered by activation of receptor interacting protein kinase 3 (RIPK3), which phosphorylates and activates the mixed lineage kinase-like domain pseudokinase, MLKL, to rupture or permeabilize the plasma membrane. Necroptosis is an inflammatory pathway associated with multiple pathologies including autoimmunity, infectious and cardiovascular diseases, stroke, neurodegeneration, and cancer. Here, we describe protocols that can be used to characterize MLKL as the executioner of plasma membrane rupture in necroptosis. We visualize the process of necroptosis in cells using live-cell imaging with conventional and confocal fluorescence&#160;microscopy, and in fixed cells using electron microscopy, which together revealed the redistribution of MLKL from the cytosol to the plasma membrane prior to induction of large holes in the plasma membrane. We present in vitro nuclear magnetic resonance (NMR) analysis using lipids to identify putative modulators of MLKL-mediated necroptosis. Based on this method, we identified quantitative lipid-binding preferences and phosphatidyl-inositol phosphates (PIPs) as critical binders of MLKL that are required for plasma membrane targeting and permeabilization in necroptosis.

We report methods for characterization of MLKL-mediated plasma membrane rupture in necroptosis including conventional and confocal live-cell microscopy imaging, scanning electron microscopy, and NMR-based lipid binding.